Tailoring the Morphology of Monodisperse Mesoporous Silica Particles Using Different Alkoxysilanes as Silica Precursors

The hard template method for the preparation of monodisperse mesoporous silica microspheres (MPSMs) has been established in recent years. In this process, in situ-generated silica nanoparticles (SNPs) enter the porous organic template and control the size and pore parameters of the final MPSMs. Here, the sizes of the deposited SNPs are determined by the hydrolysis and condensation rates of different alkoxysilanes in a base catalyzed sol–gel process. Thus, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate (TPOS) and tetrabutyl orthosilicate (TBOS) were sol–gel processed in the presence of amino-functionalized poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate) (p(GMA-co-EDMA)) templates. The size of the final MPSMs covers a broad range of 0.5–7.3 µm and a median pore size distribution from 4.0 to 24.9 nm. Moreover, the specific surface area can be adjusted between 271 and 637 m2 g−1. Also, the properties and morphology of the MPSMs differ according to the SNPs. Furthermore, the combination of different alkoxysilanes allows the individual design of the morphology and pore parameters of the silica particles. Selected MPSMs were packed into columns and successfully applied as stationary phases in high-performance liquid chromatography (HPLC) in the separation of various water-soluble vitamins.


Introduction
The introduction of high-performance liquid chromatography (HPLC) has enabled a rapid chemical analysis and separation process for substances and end products. The wide range of applications extends from small molecules [1][2][3] to pharmaceuticals [4,5], food and environmental analysis [6][7][8], long-chain polymers [9,10] and biomolecules [11][12][13]. The most common material in HPLC columns are spherical silica particles because of their mechanical robustness. Moreover, silica particles possess reactive groups on their surface, by which a variety of functionalizations allow a fine tuning of the particle features [14][15][16]. Characteristics such as particle size, dispersity, pore structure and surface functionalization influence their chromatographic properties such as selectivity, analysis time, plate number and back pressure. Due to their high specific surface area, fully porous silica particles in the µm range have proven successful in HPLC [17][18][19].
Probably the best-known representation of spherical silica networks is the silica material obtained from the Stoeber process [20]. Non-porous spherical silica particles in the

Preparation and Characterization of MPSM1a-d and MPSM2a-d
Monodisperse tetraethylenepentamine (TEPA)-functionalized p(GMA-co-EDMA) served as the template in the preparation of all MPSMs discussed here. The characteristic features of this P@TEPA template are a diameter of 6.0 ± 0.5 µm, a median pore diameter of 14.4 nm and a pore volume of 0.24 mL g −1 (Figures S2 and S3, Supporting Information).
In the presence of the P@TEPA template, a basic sol-gel process was performed with the four different alkoxysilanes, TMOS (a), TEOS (b), TPOS (c) and TBOS (d), in 2-propanol and H2O as solvents (Methods 1 and 2, Scheme 1). In Method 1, all reactants except the template are soluble in 2-propanol. Under these conditions silica nanoparticles (SNP) are formed in the continuous phase, which diffuse into the template and accumulate in the pores and on the surface [37][38][39][40]. The size of the SNPs depends on the hydrolysis and condensation rates of the precursors. With the fastest rate of hydrolysis, TMOS forms the largest SNPs, which accumulate in the pores ( Figure S4, HB1a). Moreover, the hydrolysis and condensation rates of TMOS are so high that some of the SNPs become too large (~60 nm) to enter the template network. These secondary particles remain in the continuous phase and are mostly removed from the reaction mixture during the purification process, while some of them are left at the template surface ( Figure S4, HB1a). With TEOS as the precursor, particle formation is already four times slower than for TMOS [32], resulting in smaller SNPs that easily penetrate the porous network of the template and form HB1b ( Figure S4). The longer alkoxy chains of the TPOS and TBOS alkoxides decrease their hydrolysis rates further. Thus, less silica species are available for condensation to build SNPs. The accumulation of SNPs in the template is now difficult, and SNPs are hardly observed on the surface of the hybrid particles ( Figure S4, HB1c and HB1d). Overall, the size of the SNPs decreases with the rates of hydrolysis in the TMOS > TEOS > TPOS > TBOS series ( Figure S4), and the incorporation of silica into the pores of the template is best achieved for TEOS.
In Method 2, the sol-gel process is carried out in H2O, which reduces the differences of the kinetic effects of the hydrolysis and condensation of the four different alkoxysilanes (Method 2, Scheme 1). This should have an impact on the SNP formation and the incorporation of silica into the pores of the template. Ammonia as catalyst is added after 24 h of stirring to enable the nonpolar precursors to diffuse into the template network. Condensation does not start until NH3 is added. After another 24 h, the hybrid particles HB2ad are obtained ( Figure S5 and Table 1). As a consequence of the reduced rate of the hydrolysis of TMOS, no secondary particles are observed. The particles HB2a grow by 0.7

Preparation and Characterization of MPSM1a-d and MPSM2a-d
Monodisperse tetraethylenepentamine (TEPA)-functionalized p(GMA-co-EDMA) served as the template in the preparation of all MPSMs discussed here. The characteristic features of this P@TEPA template are a diameter of 6.0 ± 0.5 µm, a median pore diameter of 14.4 nm and a pore volume of 0.24 mL g −1 (Figures S2 and S3, Supporting Information).
In the presence of the P@TEPA template, a basic sol-gel process was performed with the four different alkoxysilanes, TMOS (a), TEOS (b), TPOS (c) and TBOS (d), in 2-propanol and H 2 O as solvents (Methods 1 and 2, Scheme 1). In Method 1, all reactants except the template are soluble in 2-propanol. Under these conditions silica nanoparticles (SNP) are formed in the continuous phase, which diffuse into the template and accumulate in the pores and on the surface [37][38][39][40]. The size of the SNPs depends on the hydrolysis and condensation rates of the precursors. With the fastest rate of hydrolysis, TMOS forms the largest SNPs, which accumulate in the pores ( Figure S4, HB1a). Moreover, the hydrolysis and condensation rates of TMOS are so high that some of the SNPs become too large (~60 nm) to enter the template network. These secondary particles remain in the continuous phase and are mostly removed from the reaction mixture during the purification process, while some of them are left at the template surface ( Figure S4, HB1a). With TEOS as the precursor, particle formation is already four times slower than for TMOS [32], resulting in smaller SNPs that easily penetrate the porous network of the template and form HB1b ( Figure S4). The longer alkoxy chains of the TPOS and TBOS alkoxides decrease their hydrolysis rates further. Thus, less silica species are available for condensation to build SNPs. The accumulation of SNPs in the template is now difficult, and SNPs are hardly observed on the surface of the hybrid particles ( Figure S4, HB1c and HB1d). Overall, the size of the SNPs decreases with the rates of hydrolysis in the TMOS > TEOS > TPOS > TBOS series ( Figure S4), and the incorporation of silica into the pores of the template is best achieved for TEOS.
In Method 2, the sol-gel process is carried out in H 2 O, which reduces the differences of the kinetic effects of the hydrolysis and condensation of the four different alkoxysilanes (Method 2, Scheme 1). This should have an impact on the SNP formation and the incorporation of silica into the pores of the template. Ammonia as catalyst is added after 24 h of stirring to enable the nonpolar precursors to diffuse into the template network. Condensation does not start until NH 3 is added. After another 24 h, the hybrid particles HB2a-d are obtained ( Figure S5 and Table 1). As a consequence of the reduced rate of the hydrolysis of TMOS, no secondary particles are observed. The particles HB2a grow by 0.7 µm and HB2b by 0.3 µm and are thus larger than HB1a-d. In contrast to particles prepared by Method 1, a more edgy morphology of the hybrid materials is achieved. For TPOS (HB2c) and TBOS (HB2d) as precursors, there are no changes in size and morphology compared to the template. The thermal degradation behavior of the hybrid beads HB1a-d and HB2a-d compares well with that reported earlier ( Figure 1) [41]. After the loss of surface water, the degradation processes of the polymer backbone led to a complete decomposition of the template and allowed the determination of the silica content of the hybrid beats. Here the hybrid particles HB1a contain the highest quantity of silica (37.8 %). The amounts of silica of HB1b (29.9%), HB1c (17.7%) and HB1d (6.6%) correlate with their decreasing hydrolysis rates. The amounts of SiO 2 in HB2a and HB2b (32.7% and 35.8%, respectively) differ little ( Figure 1). Due to the suppressed hydrolysis in H 2 O, the hydrolysis rates of TMOS and TEOS are comparable. Thus, similar amounts of SiO 2 are deposited. The percentage of incorporated silica in the hybrid particles correlates well with the particle size of the resulting MPSMs (Table 1). Thermogravimetric analyses of HB2c and HB2d result in only very small amounts of SiO 2 . This is traced back to the poor miscibility of the alkoxysilanes TPOS and TBOS with water. Thus, only small amounts of SNPs are generated during the reaction. The calcination of the hybrid beads HB1a-d and HB2a-d for 10 h at 600 °C removed the organic polymer template and released the monodisperse mesoporous silica microspheres MPSM1a-d ( Figure 2) and MPSM2a-d ( Figure 3). The nanoparticulate morphology of the MPSMs is comparable to that of their corresponding hybrid beads. The particle The calcination of the hybrid beads HB1a-d and HB2a-d for 10 h at 600 • C removed the organic polymer template and released the monodisperse mesoporous silica microspheres MPSM1a-d ( Figure 2) and MPSM2a-d ( Figure 3). The nanoparticulate morphology of the MPSMs is comparable to that of their corresponding hybrid beads. The particle size of the MPSMs decreases with the decreasing hydrolysis rate of the precursors. Thus, while MPSM1a (6.0 µm) and MPSM1b (5.5 µm) represent the size of the template quite well, the sizes of MPSM1c (3.6 µm) and MPSM1d (2.2 µm) are strongly reduced. Consequently, only TMOS and TEOS map the template to 100% and 92%, respectively, while, for TPOS and TBOS, the template is mapped to only 60% and 37%, respectively. The particle sizes of MPSM2a and MPSM2b are 5.9 µm and 6.0 µm, respectively, and completely replicate the template. For MPSM2c and MPSM2d, 800 nm and 500 nm polydisperse porous silica particles are generated. The calcination of the hybrid beads HB1a-d and HB2a-d for 10 h at 600 °C removed the organic polymer template and released the monodisperse mesoporous silica microspheres MPSM1a-d ( Figure 2) and MPSM2a-d ( Figure 3). The nanoparticulate morphology of the MPSMs is comparable to that of their corresponding hybrid beads. The particle size of the MPSMs decreases with the decreasing hydrolysis rate of the precursors. Thus, while MPSM1a (6.0 µm) and MPSM1b (5.5 µm) represent the size of the template quite well, the sizes of MPSM1c (3.6 µm) and MPSM1d (2.2 µm) are strongly reduced. Consequently, only TMOS and TEOS map the template to 100% and 92%, respectively, while, for TPOS and TBOS, the template is mapped to only 60% and 37%, respectively. The particle sizes of MPSM2a and MPSM2b are 5.9 µm and 6.0 µm, respectively, and completely replicate the template. For MPSM2c and MPSM2d, 800 nm and 500 nm polydisperse porous silica particles are generated.  The pore properties of the MPSMs were determined via nitrogen adsorption/desorption measurements and are listed in Table 1. The corresponding pore size distributions are shown in Figure 4. Here, the median pore size of the MPSMs decreases and the specific surface area increases with the decreasing hydrolysis rates of the precursors. This result is consistent with the size of the SNPs that form the silica network. Large SNPs generate large pores of the MPSMs, while small SNPs result in smaller pores. [38,40] Therefore, the median pore size becomes smaller in the order of MPSM1a (23.6 nm), MPSM1b (11.3 nm), MPSM1c (8.8 nm), and MPSM1d (4.0 nm). As smaller pores form larger specific surface The pore properties of the MPSMs were determined via nitrogen adsorption/desorption measurements and are listed in Table 1. The corresponding pore size distributions are shown in Figure 4. Here, the median pore size of the MPSMs decreases and the specific surface area increases with the decreasing hydrolysis rates of the precursors. This result is consistent with the size of the SNPs that form the silica network. Large SNPs generate large pores of the MPSMs, while small SNPs result in smaller pores [38,40]. Therefore, the median pore size becomes smaller in the order of MPSM1a (23.6 nm), MPSM1b (11.3 nm), MPSM1c (8.8 nm), and MPSM1d (4.0 nm). As smaller pores form larger specific surface areas, the highest specific surface area is obtained for MPSM1d, and the lowest specific surface area is obtained for MPSM1a. The sol-gel process according to Method 2 leads to an edgier morphology for MPSM2a and MPSM2b, resulting in larger surface areas compared to MPSM1a and MPSM1b. The pore volume of the MPSMs differs between 0.5 mL g −1 and 0.9 mL g −1 . The pore properties of the MPSMs were determined via nitrogen adsorption/desorption measurements and are listed in Table 1. The corresponding pore size distributions are shown in Figure 4. Here, the median pore size of the MPSMs decreases and the specific surface area increases with the decreasing hydrolysis rates of the precursors. This result is consistent with the size of the SNPs that form the silica network. Large SNPs generate large pores of the MPSMs, while small SNPs result in smaller pores. [38,40] Therefore, the median pore size becomes smaller in the order of MPSM1a (23.6 nm), MPSM1b (11.3 nm), MPSM1c (8.8 nm), and MPSM1d (4.0 nm). As smaller pores form larger specific surface areas, the highest specific surface area is obtained for MPSM1d, and the lowest specific surface area is obtained for MPSM1a. The sol-gel process according to Method 2 leads to an edgier morphology for MPSM2a and MPSM2b, resulting in larger surface areas compared to MPSM1a and MPSM1b. The pore volume of the MPSMs differs between 0.5 mL g −1 and 0.9 mL g −1 .

Preparation and Characterization of MPSM1e and MPSM1f
The properties of the MPSMs are controlled by the hydrolysis rate of the precursors and the solvent medium. TMOS produces non-porous secondary particles while TPOS does not fully map the size of the template if the sol-gel process is carried out in 2-propanol and H 2 O. To avoid this unwanted behavior, the two precursor combinations of TMOS with TEOS (MPSM1e) and TPOS with TEOS (MPSM1f) were applied in a sol-gel process in the presence of a P@TEPA template with a diameter of 7.2 µm. The new HBs and MPSMs are shown in Figure 5. Interestingly, no secondary particles are observed for HB1e and MPSM1e. The HB1e particles have the highest silica content of all hybrid particles, and the corresponding silica microspheres have a nanoparticulate surface and exhibit a size of 7.3 µm (Table 1). Thus, they completely map the template without the negative effects of the high hydrolysis rate of TMOS. With a median pore size of 16.6 nm, this is in between that of MPSM1a and MPSM1b. This results in SNPs in the continuous phase that are smaller than those of MPSM1a and larger than those of MPSM1b. The combination of TEOS and TPOS leads to the particles HB1f and MPSM1f. The resulting silica materials have a size of 6.6 µm, representing 92% of the template. Interestingly, the median pore size of 15.6 nm and the pore volume of 1.06 mL g −1 are larger than the pore properties of MPSM1b, for which only TEOS was used. Compared with MPSM1c, the template is better replicated in MPSM1f.
are smaller than those of MPSM1a and larger than those of MPSM1b. The combination of TEOS and TPOS leads to the particles HB1f and MPSM1f. The resulting silica materials have a size of 6.6 µm, representing 92% of the template. Interestingly, the median pore size of 15.6 nm and the pore volume of 1.06 mL g −1 are larger than the pore properties of MPSM1b, for which only TEOS was used. Compared with MPSM1c, the template is better replicated in MPSM1f.

Chromatographic Measurements of MPSM1b
For the use of MPSMs as a stationary phase in high-performance liquid chromatography, high monodispersity is required to achieve efficient separation. MPSM1b particles were chosen based on their particle size and monodispersity to investigate their suitability as a stationary phase in HPLC. Therefore, MPSM1b particles were functionalized with trimethoxy (octadecyl) silane and packed in a 250 mm × 4.6 mm stainless steel column with acetone as the slurry and methanol/water (85 v.%/15 v.%) as the pressure medium.
The reproducibility of the synthesis of MPSM1b in its chromatographic properties is shown in Figure 6. The particles of three different batches with the same reaction conditions were packed in 250 mm × 4.6 mm stainless steel columns and examined for their chromatographic properties. As can be seen in Figure 6, the particles of all three batches show the same retention behavior of the test mixture. Moreover, even after one hundred injections, the retention times of toluene and uracil did not change (Supporting Information Table S1). This indicates the good stability of the stationary phase MPSM1b-C18.

Chromatographic Measurements of MPSM1b
For the use of MPSMs as a stationary phase in high-performance liquid chromatography, high monodispersity is required to achieve efficient separation. MPSM1b particles were chosen based on their particle size and monodispersity to investigate their suitability as a stationary phase in HPLC. Therefore, MPSM1b particles were functionalized with trimethoxy (octadecyl) silane and packed in a 250 mm × 4.6 mm stainless steel column with acetone as the slurry and methanol/water (85 v.%/15 v.%) as the pressure medium.
The reproducibility of the synthesis of MPSM1b in its chromatographic properties is shown in Figure 6. The particles of three different batches with the same reaction conditions were packed in 250 mm × 4.6 mm stainless steel columns and examined for their chromatographic properties. As can be seen in Figure 6, the particles of all three batches show the same retention behavior of the test mixture. Moreover, even after one hundred injections, the retention times of toluene and uracil did not change (Supporting Information  Table S1). This indicates the good stability of the stationary phase MPSM1b-C 18 .
The successful separation of five water-soluble vitamins is shown in Figure 7. A gradient from eluent A, consisting of water containing 0.025% TFA, to eluent B, consisting of acetonitrile (ACN), was used for the separation. An initial isocratic step for five minutes with eluent A is followed by an increase from eluent B to eluent A to 25/75 (v.%/v.%) in six minutes, as proposed by Heudi et al. [42]. This is followed by a second gradient on eluent B to eluent A 40/60 (v.%/v.%) in eight minutes, holding this for an additional minute. Then, the initial conditions are restored in one minute and equilibrated for four minutes. The vitamins were baseline separated and assigned based on single measurements of the analytes. The elution order is vitamin B 1 < B 3 < B 5 < B 9 < B 12 as detected at 210 nm. The successful separation of five water-soluble vitamins is shown in Figure 7. A gradient from eluent A, consisting of water containing 0.025% TFA, to eluent B, consisting of acetonitrile (ACN), was used for the separation. An initial isocratic step for five minutes with eluent A is followed by an increase from eluent B to eluent A to 25/75 (v.%/v.%) in six minutes, as proposed by Heudi et al. [42]. This is followed by a second gradient on eluent B to eluent A 40/60 (v.%/v.%) in eight minutes, holding this for an additional minute. Then, the initial conditions are restored in one minute and equilibrated for four minutes. The vitamins were baseline separated and assigned based on single measurements of the analytes. The elution order is vitamin B1 < B3 < B5 < B9 < B12 as detected at 210 nm.

Characterization
For the evaluation of the morphology, particle size and dispersity, SEM images were acquired using a Hitachi SU8030 (Krefeld, Germany). The mean particle diameter was obtained by calculating at least 400 particles from SEM images and is expressed in µm. The pore parameters of the materials are determined by nitrogen adsorption on a BELSORP MiniX from Microtrac Retsch GmbH (Haan, Germany). The sample preparation was carried out on a BELSORP VACII (Microtrac Retsch GmbH, Haan, Germany). For that, the silica materials were heated for 3 h at 300 • C, and a vacuum of 2 × 10 −2 mbar was used to remove possible physisorbed residues and to achieve a reproducible equilibrium [43]. Adsorption and desorption isotherms were performed at 77 K. For the determination of the specific surface area, the adsorption isotherms were evaluated by the Brunauer-Emmet-Teller (BET) method, and for the pore volume (single point measurement at p/p 0 = 0.95) and pore size distributions, the desorption isotherms were evaluated by the Barrett-Joyner-Halenda (BJH) method using BELMaster 7 software [44,45]. The amount of SiO 2 was determined after thermogravimetric measurements on a Mettler Toledo TGA/DSC. Samples were weighed in an aluminum vessel and measured at a heating rate of 5 K min −1 and synthetic air (50 mL min −1 ).
Analytical high-performance liquid chromatography of water-soluble vitamins was performed on an Agilent 1100 series system from Agilent Technologies (Waldbronn, Germany), which consisted of a quaternary pump with degasser, an autosampling system, a column oven and a diode array detector. Instrument control, data acquisition and automated data analysis was performed by the OpenLAB CDS (Rev. C.01.07 SR3 software, Agilent Technologies, Walbronn, Germany). A running gradient of eluent A consisting of water and 0.025 v.% TFA to eluent B consisting of acetonitrile was used according to Heudi et al. [42] The vitamins B 1 , B 5 and B 12 (1 mg mL −1 ), B 3 (0.5 mg mL −1 ) and B 9 (2 mg mL −1 ) were dissolved in water.
The hybrids HB1e-f were produced after 2.4 mL of TEOS and 1.5 mL of TMOS (e) or TPOS (f) and 0.2 mL of an aqueous ammonia solution (28-30%) were added to a dispersion of 1 g P@TEPA particles in 60 mL of 2-propanol and 7.5 mL of H 2 O. The mixture was stirred at 200 rpm for 24 h (Table 1).
Method 2: An amount of 1 g of P@TEPA particles was dispersed in 67.5 mL of H 2 O. Then, 2.4 mL of the corresponding alkoxysilane was added, and the mixture was stirred at 200 rpm. After 24 h, 0.2 mL of an aqueous ammonia solution (28-30%) was added, and the reaction was stirred for further 24 h at 200 rpm to produce hybrid beads HB2a-d ( Table 1).
All hybrid beads were separated from their solutions, washed three times with EtOH and three times with H 2 O, and dried at 65 • C for 16 h. The resulting hybrid beads were calcinated at 600 • C for 10 h to provide the corresponding mesoporous silica microspheres MPSMs (Table 1).

Octadecyl Functionalization of Mesoporous Silica Microspheres for Chromatographic Measurements
An amount of 5 g of silica particles MPSM1b was dispersed in 600 mL of hydrochloric acid (3.7%) and stirred for 3 h at 100 • C (200 rpm). The particles were separated from the solution, washed with EtOH and H 2 O until neutral and dried at 65 • C for 16 h. The particles were then dispersed in 75 mL of toluene; 25 mL of ODTMS and 0.5 mL of triethylamine were added; and the mixture was stirred at 100 • C (200 rpm) for 6 h. The particles were separated from the solution; washed three times with toluene, three times with EtOH and twice with MeOH; and dried at 65 • C for 16 h.
The functionalized particles were packed with acetone as slurry and MeOH/H 2 O (85 v.%/15 v.%) as pressure medium.

Conclusions
Monodisperse mesoporous silica microspheres (MPSM) can be tailored in their sizes and pore parameters via the hard template method. This is achieved if, at the stage of the hybrid bead syntheses, the sol-gel parameters are adjusted properly. This has been successfully demonstrated here by applying a basic sol-gel process with four different alkoxysilanes in the presence of functionalized p(GMA-co-EDMA) as the template. The SNPs grow at various rates and are thus incorporated into the template pores in nonuniform sizes, which is a consequence of the different hydrolysis and condensation rates of the alkoxysilane precursors. Thus, different amounts of silica are incorporated into the template, which has an impact on the final size of the MPSM. With TMOS and TEOS as precursors, the size of the template is reproduced, while TPOS and TBOS as precursors lead to much smaller MPSMs. Moreover, the various sizes of the incorporated SNPs generate different pore parameters. The larger the SNP, the larger the pores of the MPSM, which is important for HPLC applications. The silica particles synthesized with TEOS according to Method 1 were functionalized with trimethoxy (octadecyl) silane and used as the stationary phase in HPLC. The complete baseline separation of five water-soluble vitamins was achieved with these microspheres. The robustness of the synthesis of MPSMs in their chromatographic properties was demonstrated via HPLC using three different batches with a reversed phase test mixture.  Data Availability Statement: Data will be made available on request.